Base analogues

ABSTRACT

Nucleoside analogues or base analogues having structure (I), where X=O or NH or S, Y=N or CHR 6  or CR 6 , W=N or NR 6  or CHR 6  or CR 6  or S, n=1 or 2; each R 6  is independently H or O or alkyl or alkenyl or alkoxy or aryl or a reporter moiety; where necessary (i.e. when Y and/or W is N or CR 6  where R 6  is not O) a double bond is present between Y and W or W and W, and Q is H or a sugar or a sugar analogue or a nucleic acid backbone or backbone analogue.

This application is a divisional of U.S. Ser. No. 09/463,501, filed Apr.18, 2000, which issued on Sep. 3, 2002 as U.S. Pat. No. 6,444,682, andwhich is a 371 of PCT/GB98/02306 filed Jul. 31, 1998, which claimspriority to GB 9716231.7, filed Jul. 31, 1997.

This invention concerns base analogues which may be used to makenucleoside analogues and nucleotide analogues which may be incorporatedinto nucleic acids and nucleic acid analogues e.g. PNA. Some of theseanalogues are base-specific and may be incorporated into DNA or RNA orPNA in the place of a single native base i.e. A, T, G, or C. Otheranalogues have the potential for base-pairing with more than one nativebase or base analogue.

The present invention provides a compound having the structure

where

X=O or NH or S

Y=N or CHR⁶ or CR⁶ or CO

W=N or NR⁶ or CHR⁶ or CR⁶ or S or CO

n=1 or 2 or 3

each R⁶ is independently H or alkyl or alkenyl or alkoxy or aryl or areporter moiety,

where necessary (i.e. when Y and/or W is N or CR⁶) a double bond ispresent between Y and W or W and W, and

Q is H or a sugar or a sugar analogue,

provided that

i) when n is 2 and X is NH and W is CHR⁶ or CR⁶, and Y is CO, then atleast one reporter moiety is present,

ii) when n is 1 and X is NH and W is N or NR⁶, then at least onereporter moiety is present,

iii) when n is 1 and X is 0 and Y is CHR⁶ or CR⁶ and W is CHR⁶ or CR⁶,then at least one R⁶ is a reporter moiety which is a reactive group orsignal moiety or solid surface joined to the remainder of the moleculeby a linker of at least 3 chain atoms,

iv) when n is 1 and X is NH and Y is CHR⁶ or CR⁶ and W is CHR⁶ or CR⁶,then a least one reporter moiety is present,

v) when W is S, then n is 2 and W_(n) is —CHR⁶—S— or ═CR⁶—S—,

vi) when n is 2 and X is NH and Y is CHR⁶ or CR⁶, then at least one R⁶is a reporter moiety which is a reactive group or signal moiety or solidsurface joined to the remainder of the molecule by a linker of at least3 chain atoms.

Q may be

where

Z is O, S, Se, SO, NR⁹ or CH₂,

R¹, R², R³ and R⁴ are the same or different and each is H, OH, F, NH₂,N₃, O-hydrocarbyl or a reporter moiety,

R⁵ is OH, SH or NH₂ or mono-, di- or tri-phosphate or -thiophosphate, orcorresponding boranophosphate,

or one of R² and R⁵ is a phosphoramidite or other group forincorporation in a polynucleotide chain, or a reporter moiety,

or Q consists of one of the following modified sugar structures

 or Q is a nucleic acid backbone consisting of sugar-phosphate repeatsor modified sugar-phosphate repeats (e.g. LNA) (Koshkin et al, 1998,Tetrahedron 54, 3607-30) or a backbone analogue such as peptide orpolyamide nucleic acid (PNA). (Nielsen et al, 1991, Science 254,1497-1500).

When Q is H, these compounds are base analogues. When Q is a sugar orsugar analogue or a modified sugar, these compounds are nucleotideanalogues or nucleoside analogues. When Q is a nucleic acid backbone ora backbone analogue, these compounds are herein called nucleic acids orpolynucleotides.

Preferred general structures covered by the invention are

In the context of this invention, a nucleotide is a naturally occurringcompound comprising a heterocyclic base and a backbone including aphosphate. A nucleoside is a corresponding compound in which a backbonephosphate may or may not be present. Nucleotide analogues and nucleosideanalogues are analogous compounds having different bases and/ordifferent backbones. A nucleoside analogue is a compound which iscapable of forming part of a nucleic acid (DNA or RNA or PNA) chain, andis there capable of base-pairing with a base in a complementary chain orbase stacking in the appropriate nucleic acid chain. A nucleosideanalogue may be specific, by pairing with only one complementarynucleotide; or degenerate, by base pairing with more than one of thenatural bases, e.g. with pyrimidines (T/C) or purines (A/G); oruniversal, by pairing with each of the natural bases with littlediscrimination; or it may pair with another analogue or itself.

In one preferred aspect of the invention, the base analogue is linked toa sugar moiety such as ribose or deoxyribose to form a nucleosideanalogue. When the group R⁵ is triphosphate, the nucleoside triphosphateanalogues of the invention are capable of being incorporated byenzymatic means into nucleic acid chains.

Preferably n is 1 or 2, and W is N or NR⁶ or CR⁶ or CHR⁶.

In another preferred aspect, the nucleoside analogue or nucleotideanalogue which contains a base analogue as defined is labelled with atleast one reporter moiety. A reporter moiety may be any one of variousthings. It may be a radioisotope by means of which the nucleosideanalogue is rendered easily detectable, for example 32-P or 33-P or 35-Sincorporated in a phosphate or thiophosphate or phosphoramidite orH-phosphonate group, or alternatively 3-H or 14-C or an iodine isotope.It may be an isotope detectable by mass spectrometry or NMR. It may be asignal moiety e.g. an enzyme, hapten, fluorophore, chromophore,chemiluminescent group, Raman label or electrochemical label. Thereporter moiety may comprise a signal moiety and a linker group joiningit to the remainder of the molecule, which linker group may be a chainof up to 30 carbon, nitrogen, oxygen and sulphur atoms, rigid orflexible, unsaturated or saturated as well known in the field. Thereporter moiety may comprise a solid surface and a linker group joiningit to the rest of the molecule. Linkage to a solid surface enables theuse of nucleic acid fragments containing nucleoside analogues to be usedin assays including bead based probe assays or assays involving arraysof nucleic acid samples or oligonucleotides which are interrogated withe.g. oligonucleotide or nucleic acid or even peptide or protein probes.The reporter moiety may consist of a linker group with a terminal orother reactive group, e.g. NH₂, OH, COOH, CONH₂ or SH, by which a signalmoiety and/or solid surface may be attached, before or afterincorporation of the nucleoside analogue in a nucleic acid chain.

To avoid risk of steric hindrance, a linker preferably has at leastthree chain atoms, e.g. —(CH₂)_(n)— where n is at least 3.

Two (or more) reporter moieties may be present, e.g. a signal moiety anda solid surface, or a hapten and a different signal moiety, or twofluorescent signal groups to act as donor and acceptor. Various formatsof these arrangements may be useful for separation or detectionpurposes.

Purine and pyrimidine nucleoside derivatives labelled with reportermoieties are well known and well described in the literature. Labellednucleoside derivatives have the advantage of being readily detectableduring sequencing or other molecular biology techniques.

R¹, R², R³ and R⁴ may each be H, OH, F, NH₂, N₃, O-alkyl or a reportermoiety. Thus ribonucleosides, and deoxyribonucleosides anddideoxyribonucleosides are envisaged together with other nucleosideanalogues. These sugar substituents may contain a reporter moiety inaddition to any that Might be present on the base.

R⁵ is OH, SH, NH₂ or mono-, di- or tri-phosphate or -thiophosphate orcorresponding boranophosphate. Alternatively, one of R² and R⁵ may be aphosphoramidite or H-phosphonate or methylphosphonate orphosphorothioate or amide, or an appropriate linkage to a solid surfacee.g. hemisuccinate controlled pore glass, or other group forincorporation, generally by chemical means, in a polynucleotide chain.The use of phosphoramidites and related derivatives in synthesisingoligonucleotides is well known and described in the literature.

In the new base or nucleoside analogues to which this invention isdirected, at least one reporter moiety is preferably present in the baseanalogue or in the sugar moiety or a phosphate group. Reporter moietiesmay be introduced into the sugar moiety of a nucleoside analogue byliterature methods (e.g. J. Chem. Soc. Chem. Commun. 1990, 1547-8; J.Med. Chem., 1988, 31. 2040-8). Reporters in the form of isotopic labelsmay be introduced into phosphate groups by literature methods(Analytical Biochemistry, 214, 338-340, 1993; WO 95/15395).

Nucleoside analogues of this invention are useful for labelling DNA orRNA or for incorporating in oligonucleotides or PNA. A reporter moietyis attached at a position where it does not have a significantdetrimental effect on the physical or biochemical properties of thenucleoside analogue, in particular its ability to be incorporated insingle stranded or double stranded nucleic acid.

A template containing the incorporated nucleoside analogue of thisinvention may be suitable for copying in nucleic acid synthesis. If areporter moiety of the incorporated nucleoside analogue consists of alinker group, then a signal moiety can be introduced into theincorporated nucleoside analogue by being attached through a terminal orother reactive group of the linker group.

A nucleoside analogue triphosphate of this invention may be incorporatedby enzymes such as terminal transferase to extend the 3′ end of nucleicacid chains in a non-template directed manner. Tails of the nucleosideanalogue triphosphate produced in this way may be detected directly inthe absence of any reporter label by use of antibodies directed againstthe nucleoside analogue. The analogues when incorporated intooligonucleotides or nucleic acids may be acted upon by nucleic acidmodification enzymes such as ligases or restriction endonucleases.

In primer walking sequencing, a primer/template complex is extended witha polymerase and chain terminated to generate a nested set of fragmentswhere the sequence is read after electrophoresis and detection(radioactive or fluorescent) or directly in a mass spectrometer A secondprimer is then synthesised using the sequence information near to theend of the sequence obtained from the first primer. This second(“walking”) primer is then used for sequencing the same template. Primerwalking sequencing is more efficient in terms of generating lessredundant sequence information than the alternative “shot gun” approach.

The main disadvantage with primer walking is the need to synthesise aprimer after each round of sequencing. Cycle sequencing requires primersthat have annealing temperatures near to the optimal temperature for thepolymerase used for the cycle sequencing. Primers between 18 and 24residues long are generally used for cycle sequencing. The size of apresynthesised walking primer set required has made primer walking cyclesequencing an impractical proposition. The use of base analogues thatare degenerate or universal addresses this problem. The use of suchanalogues that are also labelled, e.g. the nucleoside analogues of thisinvention will also help to overcome the problem. Preferred reportersfor this purpose are radioactive isotopes or fluorescent groups, such asare used in conventional cycle sequencing reactions. Where thenucleoside analogues are base specific chain terminators they may beused in chain terminating sequencing protocols.

The final analysis step in DNA sequencing involves the use of adenaturing polyacrylamide electrophoresis gel to separate the DNAmolecules by size. Electrophoretic separation based solely on sizerequires the complete elimination of secondary structure from the DNA.For most DNA this is typically accomplished by using high concentrationsof urea in the polyacrylamide matrix and running the gels at elevatedtemperatures. However certain sequences, for example those capable offorming “stem loop” structures retain secondary structure and, as aresult, display compression artefacts under standard electrophoresisconditions. Here, adjacent bands of the sequence run at nearly the sameposition on the gel, “compressed” tightly together. Such loops aretypically formed when a number of GC pairs are able to interact since GCpairs can form 3 hydrogen bonds compared to the 2 hydrogen bonds of ATpairs.

A second form of compression artefact is seen when rhodamine-labelledterminators are used and there is a G residue close to the terminus. Inthese cases, anomalous mobility of the DNA strand in a gel is oftenseen, possibly due to an interaction between the dye and the G residue.

Thus, compression artefacts appear to be caused whenever stablesecondary structures exist in the DNA under the conditions prevailing inthe gel matrix during electrophoresis. The folded structure runs fasterthrough the gel matrix than an equivalent unfolded DNA.

Currently, gel compression artefacts are eliminated in one of two ways.One is to change to a stronger denaturing condition for the gel, forexample 40% formamide with 7 M urea. The other method is to incorporatea derivative of dGTP during the synthesis of DNA. An alternative methodwould involve the use of a dCTP analogue which reduced the hydrogenbonding potential of the G-C base pair. The nucleoside analogues of thisinvention may be useful in this regard.

The nucleoside analogues of this invention can also be used in any ofthe existing applications which use native nucleic acid probes labelledwith haptens, fluorophores or other reporter groups, for example onSouthern blots, dot blots and in polyacrylamide or agarose gel basedmethods or solution hybridization assays and other assays in microtitreplates or tubes or assays of oligonucleotides or nucleic acids such ason microchips. The probes may be detected with antibodies targetedeither against haptens which are attached to the base analogues oragainst the base analogues themselves which would be advantageous inavoiding additional chemical modification. Antibodies used in this wayare normally labelled with a detectable group such as a fluorophore oran enzyme. Fluorescent detection may also be used if the base analogueitself is fluorescent or if there is a fluorophore attached to thenucleoside analogue.

The use of the different mass of the nucleoside analogue may also beused as a means of detection as well as by the addition of a specificmass tag identifyer to it. Methods for the analysis and detection ofspecific oligonucleotides, nucleic acid fragments and oligonucleotideprimer extension projects based on mass spectrometry have been reported.(Beavis R. C., Chait B. T., U.S. Pat. No. 5,288,644, Wu K. J. et al.Rapid Commun. Mass Spectrom. 7,142 (1993), Koster H. WO 94/16101.

These methods are usually based on matrix assisted laser ionisation anddesorption, time of flight (MALDITOF) mass spectrometry. They measurethe total mass of an oligonucleotide or fragment and from this thesequence of the specific oligonucleotide may be able to be ascertained.In some cases the mass of the oligonucleotide or fragment may not beunique for a specific sequence. This will occur when the ratio of thefour natural bases, ACGT, is similar in different sequences.

For example, a simple 4 mer oligonucleotide, ACGT will have the samemass as 24 other possible mers, for example; CAGT, CATG, CGTA, CTAG,CTGA etc.

With longer nucleic acid fragments it may be difficult to resolve thedifferences in mass between 2 fragments because of a lack of resolutionin the spectrum at higher molecular weights. The incorporation of theanalogues described here can be used to help identify the specificoligonucleotide or nucleic acid fragment as their masses are differentfrom those of the natural bases.

For example, the two sequences ACGT and CAGT can be identified in thepresence of one another by mass spectrometry if, for example one of thenatural nucleotides in one of the sequences is replaced with one of theanalogues described in this invention. For example, in theoligonucleotide CAGT the T can be replaced by an analogue with littleeffect on a specific application, for example hybridisation or enzymaticincorporation. Yet the two sequences can be readily identified in themass spectrometer because of the change in mass due to the introductionof the analogue base.

Not only can mass modifications be made to the bases or linkers but alsoto the sugars or inter nucleotide linkages. For example thio sugars orphosphorothioate linkages will also result in distinctive mass changes.

A mixture of modifications at the base, linker, nucleoside or nucleotideeither separately or together can give rise to a number of moleculeswith different masses which will be useful to define a specific sequenceaccurately by its mass, especially in multiplex nucleic acidhybridisation or sequencing applications.

The nucleoside analogues of the present invention with the combinationof molecular diversity and increased numbers of positions where reportergroups may be added can result in a series of improved enzymesubstrates.

Another preferred aspect of the invention is to incorporate thenucleoside analogue triphosphate into DNA by means of a polymerase butwithout a reporter label for the purpose of random mutagenesis. It hasbeen shown by Zaccolo et al, 1996, J. Mol. Biol. 255, 589-603 that whennucleotide analogues with ambivalent base pairing potential areincorporated by the PCR into DNA products, they induce the formation ofrandom mutations within the DNA products. In the above publication, thenucleotide analogue dPTP was shown to be incorporated into DNA by Taqpolymerase in place of TTP and, with lower efficiency, dCTP. After 30cycles of DNA amplification, the four transition mutations A→G, T→C, G→Aand C→T were produced. The compound 8-oxodGTP was also used to cause theformation of the transversion mutations A→C and T→G. The nucleosideanalogue triphosphates with ambivalent base pairing potential describedwithin this invention may be used for a similar purpose.

RNA is an extremely versatile biological molecule. Experimental studiesby several laboratories have shown that in vitro selection techniquescan be employed to isolate short RNA molecules from RNA libraries thatbind with high affinity and specificity to proteins, not normallyassociated with RNA binding, including a few antibodies, (Gold, Allen,Binkley, et al,1993, 497-510 in The RNA World, Cold Spring Harbor Press,Cold Spring Harbor N.Y., Gold, Polisky, Unlenbeck, and Yarus, 1995,Annu. Rev. Biochem. 64: 763-795, Tuerk and Gold, 1990, Science249:505-510, Joyce, 1989, Gene 82:83-87, Szostak, 1992, Trends Biochem.Sci 17:89-93, Tsai, Kenan and Keene, 1992, PNAS 89:8864-8868, Tsai,Kenan and Keene, 1992, PNAS 89:8864-8868, Doudna, Cech and Sullenger,1995, PNAS 92:2355-2359). Some of these RNA molecules have been proposedas drug candidates for the treatment of diseases like myasthenia gravisand several other auto-immune diseases.

The basic principle involves adding an RNA library to the protein ormolecule of interest. Washing to remove unbound RNA. Then specificallyeluting the RNA bound to the protein or other molecule of interest. Thiseluted RNA is then reverse transcribed and amplified by PCR. The DNA isthen transcribed using modified nucleotides (either 2′ modifications togive nuclease resistance e.g. 2′ F, 2′ NH₂, 2′ OCH₃ and/or C5 modifiedpyrimidines and/or C8 modified purines). Those molecules that are foundto bind the protein or other molecule of interest are cloned andsequenced to look for common (“consensus”) sequences. This sequence isoptimised to produce a short oligonucleotide which shows improvedspecific binding which may then be used as a therapeutic.

The base analogues described here, when converted to the deoxy- orribonucleoside triphosphate or deoxy- or ribonucleoside phosphoramidite,will significantly increase the molecular diversity available for thisselection process. This may lead to oligonucleotides with increasedbinding affinity to the target that is not available using the currentbuilding blocks.

The secondary structure of nucleic acids is also important whenconsidering ribozyme function. The base analogues of the presentinvention may cause the formation of secondary structures which wouldotherwise be unavailable using native bases or other modified nucleotidederivatives.

The hybridization binding properties of nucleic acids incorporating baseanalogues of the present invention may have particular application inthe antisense or antigene field.

The base analogues of the present invention may have properties whichare different to those of the native bases and therefore areparticularly suited to other important applications. In particular, theinteraction of these base analogues with enzymes may be extremelyimportant in vivo and may result in the development of new anti-viraltherapeutics or therapeutics for non-viral diseases.

A wide range of nucleoside and nucleotide analogues have been developedto form an original class of antiviral agents. Some of these compoundshave already been approved by the US FDA for use in the treatment ofviral diseases. Examples are compounds like 3′-deoxy-3′-azidothymidine(AZT, Zidovudine) and 2′,3′-dideoxy-3′-thiacytidine (3TC, Lamivudine)for the treatment of HIV infections. Other compounds like(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC, cidofovir),9-(2-phosphonyl-methoxyethyl)adenine (PMEA, adefovir) and(R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA), the acyclic nucleosidephosphonate analogues, are in clinical trials. These compounds eitheract as absolute DNA chain terminators or result in termination afterincorporation of consecutive molecules causing inhibition of the viralDNA polymerase. It should be noted that some of these compounds are theunnatural β-L enantiomers which show significantly decreased interactionwith the host DNA polymerases compared to the viral polymerases.

One of the problems in the treatment of viral infections with nucleosideand nucleotide drugs is the ability of the virus to develop resistanceby a series of mutations to the viral reverse transcriptase gene thatare selected as a result of drug pressure. Therefore, it is oftennecessary to use combination drug therapies to overcome this problem.However, the number of suitable, available compounds for therapy islimited. The subject of this invention could be useful in expanding therange of nucleoside and nucleotide antiviral drugs.

Those skilled in the art of organic chemistry will recognise that thereis a variety of approaches that can be taken to the compounds claimedwithin the scope of the claims. In addition to those approaches detailedin the experimental section those illustrated below are possible.

In order to synthesise a compound where W=CR₆ and R₆ contains a reportergroup the reaction sequence in scheme 1 can be used.

Diacetyl protected deoxycytidine an be treated withethyltrifluorobutynoate to get an enamine intermediate. The enamine isexpected to undergo oxidative cyclization under Pd(OAc)₂/DMA/70° C.reaction conditions (Fukuda et al, Bioorganic & Medicinal ChemistryLetters, 1997, 7, 1683). The ester group thus incorporated can beexploited to conjugate with a fluorescent dye, e.g. after converting toa suitable functional group such as an active ester it can be eitherreacted directly with an amine containing fluorescent dye or with a witha suitably protected diamine to extend the linker group prior to signalattachment.

When X and W both equal N the following approach in scheme 2 can beundertaken

Treatment of the known 5-aminocytidine (Kalman and Goldman, BBRC, 1981,102, 682) with ethylformate leads to the product (R=H).

For the 8-oxoG analogue (i.e. R=O) treatment of the initial diamine witha variety of reagents (COCl₂, carbonyl diimidazole, diphenyl carbonate)leads to the desired product.

These can be converted to its 5′-triphosphate using methods outlined inthe experimental section and its 5′-dimethoxytrityl-3′-phosphoramiditeby standard literature methods.

The introduction of a linker (R₆) to the 8-position (conventional purinenumbering) can be carried out using the reactivity of this position tobromination followed by alkylation with, for example, hexane diamine.

Experimental Schemes

General Procedure

Ion exchange (IE) HPLC was performed on a Waters analytical systemrunning under Millenium Chromatography Manager software. For analyticalIE analysis a Amersham Pharmacia Biotech □RPC ST (C₂/C₁₈) reverse phasecolumn (4.6×100 mm) was used, with (method A) a gradient of 0-25% bufferB over 30 min at a flow rate of 1 mL/min or (method B) a gradient of0-50% buffer B over 30 min and the same flow rate as method A. Buffer Awas 0.1 M TEAB and buffer B was 100% acetonitrile TLC analysis wasperformed on 0.2 mm-thick precoated Merck silica gel 60 F₂₅₄ plates.Flash silica gel chromatography was performed with 230-400 mesh 60-Åsilica from Merck.

¹H NMR spectra (300 MHz) were recorded on a Varian Gemini system

EXAMPLE 1 Synthesis of1-(2′-deoxy-5′-triphospho-β-D-ribofuranosyl)piperidino[2,3-d]pyrimidine-2(1H)-one(1.6)

3′,5′-Di-O-p-toluoyl-5-iodo-2′-deoxyuridine (1.1)

A stirred solution of 5-iodo-2′-deoxyuridine (2.45 g, 6.92 mmol) inanhydrous pyridine (50 mL) was cooled to 0° C. and p-toluoyl chloride(2.29, 17.3 mmol, 2.5 eq) was slowly added. The solution was allowed towarm to room temperature, heated at 50° C. for 3 hrs and then stirred atroom temperature overnight. The solvent was evaporated in vacuo to givea white solid mass after repeated co-evaporation with EtOH. Chloroformwas added and the organic phase was washed with 0.5 M H₂SO₄ (2×25 mL)then water (2×25 mL), dried over anhydrous sodium sulfate, filtered andevaporated to dryness. The crude material was crystallized at −18° C. in100 mL of a mixture MeOH/CHCl₃ 5:1 to give 3.3 g of title compound (80%yield). m.p. 193° C. ¹H d₆-DMSO δ(ppm) 2.36 and 2.38 (6H, 2×s, 2×Me),2.49-2.67 (2H, m, H2′, H2″), 4.44-4.58 (3H, m, H4′, H5′, H5″), 4.50-4.55(1H, m, H3′), 6.21 (1H, t, J=6.7 Hz, H1′), 7.30-7.36 (m, 4H, aromatic),7.86-7.92 (m, 4H, aromatic), 8.08 (1H, s, H6), 11.79 (1H, brs, NH). m/z(MALDI-TOF MS) 613.6 (M+Na)+, 629.6 (M+K)+, C₂₅H₂₃IN₂O₇ requires590.372.

3′,5′-Di-O-p-toluoyl-5-N-trifluoroacetylaminopropargyl-2′-deoxyuridine(1.2)

In a three-necked flask containing 60 mL of freshly distilled DMF purgedwith nitrogen was added the nucleoside (1.1) (2.45 g, 4 mmol),N-propargyl trifluoroacetamide (1.81 g, 12 mmol, 3 eq),tetrakis-bis-triphenylphosphine-palladium(0) (115 mg, 0.1 mmol, 2.5%mmol), Cul (38 mg, 0.2 mmol, 5% mmol) and triethylamine (2.8 mL, 20mmol, 5 eq). The reaction was purged with nitrogen and the solutionstirred at room temperature overnight. After this timetetrakis-bis-triphenylphosphine-palladium(0) (115 mg, 0.1 mmol, 2.5%mmol), Cul (38 mg, 0.2 mmol, 5% mmol) were added and the stirringcontinued until TLC (EtOAc/Hexane 1:1) showed the reaction was complete(20 hrs). To the mixture solid EDTA disodium salt (0.5 g) was added,then the solvent was concentrated and diluted with 100 mL of chloroform.The solution was washed with 5% EDTA disodium salt (3×50 mL), water(2×50 mL) and brine (2×50 mL). The organic phase was dried overanhydrous sodium sulfate, filtered and evaporated to dryness. The crudebrownish solid was purified by silica gel column chromatography(MeOH/CHCl₃ 0→2%) to give 2.2 g of pure material as a yellowish solid(89% yield). The purity of the compound was assessed by analytical HPLCon RP-C18 (gradient CH₃CN/H₂O in presence of 0.1% TFA, retention time 15min). m.p. 196-198° C. with dec. ¹H-NMR (CDCl₃): δ(ppm) 2.27-2.37 (1H,m, H2′), 2.42 and 2.44 (6H, 2×s, 2×Me), 2.79-2.85 (1H, m, H2″), 4.17(2H, t, J=4.6 Hz, N—CH₂), 4.61-4.76 (3H, m, H4′, H5′, H5″), 5.62 (1H, d,J=6.2 Hz, H3′), 6.36 (1H, dd, J=5.6 and 8.0 Hz, H1′), 6.97 (1H, brs,NH—CH₂), 7.20-7.30 (m, 4H, aromatic), 7.86-7.96 (m, 5H, aromatic, H6),8.61 (1H, brs, NH). m/z (MALDI-TOF MS) 636.8 (M+Na)+, C₃₀H₂₆F₃N₃O₈requires 613.547.

3′,5′-Di-O-p-toluoyl-5-N-trifluoroacetylaminopropyl-2′-deoxyuridine(1.3)

To a solution of the nucleoside (1.2) (2 g, 3.26 mmol) in THF (130 mL),10% Pd/C (250 mg) was added and the mixture was hydrogenated (1 atm) atroom temperature with vigorous stirring. After 12 hrs the reaction wascomplete (MALDI-TOF MS indicated the saturation of the triple bond tosingle bond). The suspension was filtered through celite and the cakewashed with MeOH. The solvent was evaporated and the residue dissolvedin CHCl₃ then washed with brine. The organic layer was dried overanhydrous Na₂SO₄ then evaporated to dryness. The crude material waspurified by silica gel column chromatography (MeOH/CHCl₃ 0→2%) to give1.85 g of pure material as a white solid (92% yield). ¹H-NMR (CDCl₃): δH(ppm) 1.50-1.59 (2H, m, CH₂CH₂CH₂), 2.05-2.14 (2H, m, 5-CH₂), 2.27-2.37(1H, m, H2′), 2.42 and 2.44 (6H, 2×s, 2×Me), 2.44-2.78 (1H, m, H2″),3.17-3.24 (2H, m, N—CH₂), 4.55 (1H, brs, H5′), 4.63-4.68 (1H, m, H5″),4.81-4.86 (1H, m, H4′), 5.64 (1H, d, J=6.2 Hz, H3′), 6.36 (1H, dd, J=5.4and 8.8 Hz, H1′), 7.20-7.30 (4H, m, aromatic), 7.37 (1H, s, H6), 7.40(1H, brs, NH—CH₂), 7.90-7.97 (4H, m, aromatic), 8.34 (1H, brs, NH). m/z(MALDI-TOF MS) 640.7 (M+Na)+, 656.7 (M+K)+, C₃₀H₃₀F₃N₃O₈ requires617.579.

1-(3′,5′-Di-O-p-toluoyl-2′-deoxy-β-D-ribofuranosyl)-4-(1,2,4-triazolyl)-5-N-trifluoroacetylaminopropyl-1H-pyrimidin-2-one(1.4)

To 1,2,4-triazole (3 g, 43.6 mmol previously dried by repeatedcoevaporations with anhydrous pyridine) suspended in anhydrousacetonitrile (75 mL) at 0° C., was added dropwise phosphoryl chloride(0.81 mL, 8.7 mmol) and the mixture was stirred for 10 min at 0° C. Tothis suspension freshly distilled triethylamine (7.4 mL, 52 mmol) wasthen added and the suspension stirred for 20 min at 0° C. and for afurther 10 min at room temperature under nitrogen. After this time wasadded dropwise a solution of the nucleoside (1.3) (1.75 g, 2.91 mmol) in40 mL of anhydrous acetonitrile and stirred at overnight at roomtemperature under nitrogen. The solvent was removed and the residuepartitioned between chloroform (50 mL) and NaHCO₃ (2×30 mL), then brine(30 mL). The organic phase was dried with anhydrous sodium sulfate andthen concentrated to dryness. The crude material was purified by silicagel column chromatography (EtOAc/Hexane 1:1→8:2) to give 0.96 g ofoff-white foam (50% yield). ¹H-NMR (CDCl₃): δ(ppm) 1.57-1.69 (2H, m,CH₂CH₂CH₂), 2.28-2.37 (4H, m, H2′, Me), 2.45 (3H, s, Me), 2.67-2.80 (2H,m, 5-CH₂), 3.20-3.32 (3H, m, N—CH₂, H2″), 4.65-4.72 (2H, m, H5′, H5″),4.90-4.94 (1H, m, H4′), 5.65 (1H, d, J=6.1 Hz, H3′), 6.38 (1H, d, J=5.9Hz, H1′), 6.99 (1H, brs, NH—CH₂), 7.20-7.37 (4H, m, aromatic), 7.84 (2H,d, J=8.0, Hz aromatic), 7.98 (2H, d, J=8.0, Hz aromatic), 8.11 (1H, s,H6), 8.15 (1H, s, triazole), 9.30 (1H, s, triazole). m/z (MALDI-TOF MS)692.2 (M+Na)+, 708.3 (M+K)+, C₃₂H₃₁F₃N₆O₇ requires 668.629.

1-(2′-deoxy-β-D-ribofuranosyl)piperidino[2,3-d]pyrimidine-2(1H)-one(1.5)

The nucleoside (1.4) (0.9 g, 1.34 mmol) was dissolved in 30 mL ofmethanolic ammonia and the solution vigorously stirred at roomtemperature. After 22 hrs stirring at room temperature a small amount offinal product was detected by MALDI-TOF MS. The solvent was removed byrotary evaporation, the residue re-dissolved in MeOH and the solutionrefluxed for 1 hr (the formation of a new product having Rf 0.3 on TLCplate, run with MeOH/CHCl₃ 20%, was observed). The solvent was removedand the residue purified by silica gel column chromatography (MeOH/CHCl₃0→20%) to give 180 mg of pure compound as a white foam (50% yield).¹H-NMR (d₆-DMSO): δH (ppm) 1.67-1.72 (2H, m, CH₂CH₂CH₂), 1.90-2.07 (2H,m, H2′, H2″), 2.40-2.45 (2H, m, 5-CH₂), 3.14-3.22 (2H, m, N—CH₂),3.48-3.58 (2H, m, H5′, H5″), 3.70-3.74 (1H, m, H4′), 4.17-4.21 (1H, m,H3′), 4.98 (1H, t, J=5.1 Hz, OH), 5.17 (1H, d, J=4.0 Hz, OH), 6.16 (1H,d, J=7.0 Hz, H1′), 7.57 (1H, s, H6), 7.83 (1H, brs, NH). m/z (MALDI-TOFMS) 268.8 (M+H)+, 290.8 (M+Na)+, 306.7 (M+K)+, C₁₂H₁₇N₃O₄ requires267.285. UV (EtOH) λmax 286 nm (e=9.8×10³) and 203 nm (e=24.0×10³), λmin236 nm (e=4.9×10³), λsh 262 nm (e=7.4×10³) and 222 nm (e=7.1×10³); pH1λmax 297 nm (e=13.9×10³) and 219 nm (e=8.4×10³), λmin 253 nm(e=2.2×10³); pH12 λmax 281 nm (e=25.3×10³).

1-(2′-deoxy-5′-triphosphate-β-D-ribofuranosyl)piperidino[2,3-d]pyrimidine-2(1H)-one(1.6)

The nucleoside (1.5) (100 mg, 0.374 mmol) and1,8-bis(dimethylamino)naphthalene (Proton Sponge) (120 mg, 0.561 mmol,1.5 eq) were dissolved in 2.2 mL of trimethylphosphate (dried with 4 Åmolecular sieves) and the solution was cooled to −20° C. Phosphorylchloride (70 μL, 0.748 mmol, 2 eq) was added and the mixture stirredwith cooling (temperature was kept between −20 and −10° C.) for 4 hrs.After this time, to the purple solution tri-n-butylammoniumpyrophosphate (3.74 mL of 0.5 M solution in anhydrous DMF, 1.87 mmol, 5eq) was added followed by tri-n-butylamine (0.37 mL, 1.57 mmol, 4.2 eq)and the mixture was stirred at −20° C. for 3 mins. Then 25 mL of 0.4 MTEAB solution was added and the mixture stirred at room temperature for10 mins. Water and TEAB were removed by rotary evaporation and to theresidue diethyl ether (30 mL) was added. The solvent was decanted andthe gummy residue was washed once again with 30 mL of diethyl ether.After decanting the residue was dried to give 1.7 g of a gummy crudematerial. The crude material was purified by ion exchangechromatrography (0-100% B over 90 min. B=0.3M trimethylammoniumbicarbonate, 3 ml/min) then the product further purified by reversephase separation using a Waters Sep-Pak C18 cartridge eluting with 20%acetonitrile/0.1M triethylammonium bicarbonate to give the titlecompound. ¹H-NMR (D₂O) δ(ppm) 1.68-1.72 (2H, m, CH₂CH₂CH₂), 2.16 (2H, m,H2′,H2″), 2.45 (2H, m, 5-CH₂), 3.16 (2H, m, N—CH₂), 3.16 (2H, m,H5′,H5″), 4.02 (1H, m, H4′), 4.17-4.12 (1H, m, H3′), 6.07 (1H, m, H1′),7.60 (1H, s, H6). ³¹P-NMR (D₂O): δ(ppm) −10.46 (d), −11.62 (d), −23.33(t)

The nucleoside (1.7) and its triphosphate derivative can be made by ananalogous route by carrying out a Lindlar reduction on the nucleoside(1.2) and then repeating the synthetic steps above. The experimental forthe Lindlar reduction is detailed below.

Dissolve 1.5 g of (1.2) in 75 mL of methanol in a Parr hydrogenationflask. With a pasteur pippete, add 5 drops of quinoline and 0.5 g ofPd/CaCO₃ (Lindlar catalyst-Aldrich)(0.3 equivalents by weight). Put theflask to a Parr hydrogenation apparatus, evacuate the air with hydrogenthree times using a water aspirator, regulate the hydrogen pressure to30 psi and shake the flask overnight. The reaction is checked forcompletion by TLC (20% MeOH/Chloroform). When the reaction has gone tocompletion the catalyst is filtered off, wash the filter with methanoland coevaporate the filtrate with 50 g of silica gel. The product is thepurified by gradient flash chromatography using the following 10, 15,20, 25% MeOH/Chloroform which afforded 1.2 g of the required olefinicintermediate as an intermediate en route to (1.7), 70% yield.

EXAMPLE 2 Synthesis of3-(2-deoxy-β-D-erythro-pentofuranosyl)-6-(butyl-4-N-2,2,2-trifluoro-acetamide)-furano[2,3-d]pyrimidin-2-one(2.6)

3′,5′-Di-O-acetyl-5-iodo-2′-deoxyuridine (2.2)

To 5-iodo-2′-deoxyuridine (2.1) (70.8 g, 200 mmol, 1 eq) in an 500 mLErlenmeyer flask was added acetic acid (72 g, 68.7 mL, 1200 mmol, 6 eq)and acetic anhydride (51 g, 47.1 mL, 500 mmol, 2.5 eq). In a test tubeat 0° C., the catalyst for the reaction was prepared by adding HClO₄(0.5 mL) over acetic anhydride (2 mL). Five drops of the catalyst wereadded into the Erlenmeyer flask at 0° C. with stirring. The reactionmixture was brought to room temperature and stirred for 4 hours whereupon a thick white precipitate formed. The Erlenmeyer flask was filledwith ether and stirred vigorously for 30 minutes. The white precipitatewas filtered and the white cake was washed with ether (500 mL). Theproduct was dried in the vacuum oven overnight to afford (2.2) (90.1 g,95%) ¹H-NMR (CDCl₃) δ(ppm) 9.40 (bs, 1 H, exchangeable, NH), 7.78 (s, 1H, H-6), 6.18 (dd, J=8.10 Hz, 1 H, H-1′), 5.05 (m, 1 H, H-3′), 4.07-4.25(m, 3 H, H-6), 4′, 5′), 2.35 (m, 1 H, H-2′_(b)), 1.85-2.12 (m, 7 H,2×CH₃, H-2′_(a))

3′,5′-Di-O-acetyl-5-(pentyn-5-Cyano)-2′-deoxyuridine (2.3)

This compound was prepared from (2.1) (4.73 g, 10 mmol, 1 eq) in asimilar manner as (2.4) except the reaction mixture was stirredovernight at room temperature to afford (2.3) (3.04 g, 75%): UV (MeOH)λ_(max) 289 nm; ¹H-NMR (CDCl₃) δ9.27 (s, 1 H, exchangeable, H—NH), 7.80(s, 1 H, H-6), 6.30 (t, J=6.55 Hz, 1 H, H-1′), 5.25 (m, 1 H, H-3′),),4.40 (m, 3 H, H-4′, 5′), 2.60 (m, 5 H, H-2′_(β), C≡CCH₂CH₂CH₂CN), 2.10(m, 7 H, H-2′_(α), 2×CH₃), 1.95 (m, 2 H, H—CH₂CH₂CH₂). This compound canbe cyclised to (2.4) by refluxing with a catalytic amount of Cul and tenequivalents of triethylamine in methanol.

6-Butyronitrile-3-(3,5-Di-O-acetyl-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one(2.4)

Triethylamine (3.04 g, 4.2 mL, 30 mmol, 2 eq) was added to a stirredsolution of (2.2) (7.1 g, 15 mmol, 1 eq), Pd[P(C₆H₅)₃]₄ (1.73 g, 1.5mmol, 0.1 eq), Cul (0.57 g, 3 mmol, 0.2 eq) and hexynenitrile (2.79 g,30 mmol, 2 eq) in dry DMF (50 mL) and the reaction mixture was thenstirred overnight at 55° C. under an inert atmosphere. To the reactionmixture was then added the bicarbonate form of AG1×8 resin to removetriethylammonium hydroiodide, CHELEX resin to remove metal cations andactivated charcoal to remove colour. After filtration on Celite aslightly yellow solution was produced. Solvent removal under high vacuumgave a dark residue which was dissolved in methanol and coevaporatedwith silica gel (75 g). Flash chromatography with 10%methanol/chloroform as eluant afforded (2.3) (3.9 g, 64%) UV (MeOH)λ_(max) 324 nm; ¹H-NMR (CDCl₃) δ(ppm) 8.22 (s, 1 H, H-6), 6.30 (t,J=6.55 Hz, 1 H, H-1′), 6.25 (s, 1 H, HC═C(O)CH₂), 5.20 (m, 1 H, H-3′),4.40 (m, 3 H, H4′, 5′), 2.92 (m, 3H, H-2′_(b), HC═C(O)CH₂CH₂), 2.43 (t,2 H, H—CH₂CH₂CN), 2.05-2.10 (m, 9 H, H-2′_(a), 2×CH₃, CH₂CH₂CH₂)

6-Butyronitrile-3-(2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one(2.5)

To 2 M KOH solution (1.6 mL) was added a solution of (2.4) (0.61 g, 1.5mmol) in methanol (10 mL) and the mixture stirred for 4 minutes. TLC(20% methanol/chloroform) showed the reaction had gone completion. Afterneutralization with 1 N HCl the solvents were removed under reducedpressure. The resulting residue was dissolved in methanol and anyremaining insoluble salt filtered out. Removal of the solvent providedcrude (2.5) (0.48 g, 97%) UV (MeOH) λ_(max) 324 nm; ¹H-NMR (d₆-DMSO)δ(ppm) 8.75 (s, 1H, H-6), 6.57 (s, 1 H, H—CH═C(O)CH₂), 6.30 (t, J=6.55Hz, 1 H, H-1′), 5.33 (m, 1 H, exchangeable, H—OH), 5.18 (m, 1 H, H—OH),4.23 (m, 1 H, H-3′), 3.92 (m, 1 H, H4′), 3.68 (m, 2 H, H-5′), 2.80 (t,J=, 2 H, H—CH═C(O)CH₂CH₂), 2.60 (t, 2H, H—CH₂CH₂CN), 2.40 (m, 2 H,H-2′_(b)), 2.05 (m, 1 H, H-2′_(a)), 1.92 (m, 2 H, H—CH₂CH₂CH₂CN)

2,2,2-Trifluoro-N-(3-(2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one-6-Butyl)-acetamide(2.6)

A solution of (2.5) (0.1 g, 0.32 mmol) in methanol (15 mL) and Raney-Nihydrogenation catalyst (1 g) were loaded into a Parr apparatus andstirred under hydrogen (p=25 psi) for 4 hours. TLC (20%methanol/chloroform) shows reaction completion. The reaction mixture wasfiltered through Celite. To the filtrate was then added ethyltrifluoroacetate (1 mL) and triethylamine (0.5 mL) and the mixture leftto stirr for 4 hours. Solvent evaporation and chromatography (15-20%methanol/chloroform) afforded (2.6) (81 mg, 60%) UV (MeOH) λ_(max) 324nm; ¹H-NMR (d₆-DMSO) δ(ppm) 9.40 (bs, 1 H, exchangeable, H—NHCOCF₃) 8.75(s, 1 H, H-6), 6.47 (s, 1 H, H—CH═C(O)CH₂), 6.18 (t, J=6.55 Hz, 1 H,H-1′), 5.33 (m, 1 H, exchangeable, H—OH), 5.18 (m, 1 H, H—OH), 4.23 (m,1 H, H-3′), 3.92 (m, 1 H, H4′), 3.68 (m, 2 H, H-5′),), 3.20 (m, 2 H,H—CH₂CH₂NH), 2.62 (t, J=, 2 H, H—CH═C—O—CH₂CH₂), 2.40 (m, 2 H,H-2′_(b)), 2.05 (m, 1 H, H-2′_(a)), 1.60-1.80 (m, 4 H, H—CH₂CH₂CH₂CH₂NH)

EXAMPLE 3

Synthesis of6-methylamine-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)(3.3a) and5-Carboxyfluoresceinyl-(6-methylamine-3-(5-triphosphate-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)(3.3b)

2,2,2-Trifluoro-N-(3-(2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one-6-methyl)-acetamide(3.1a)

Triethylamine (3.04 g, 4.2 mL, 30 mmol, 1.5 eq) was added to a stirredsolution of 5-iodo-2′-deoxyuridine (7.1 g, 20 mmol, 1 eq), Pd[P(C₆H₅)₃]₄(1.16 g, 1 mmol, 0.05 eq), Cul (0.38 g, 2 mmol, 0.1 eq) and2,2,2-trifluoro-N-propargyl-acetamide (4.53 g, 30 mmol, 1.5 eq) in dryDMF (75 mL) and the resulting reaction mixture was then stirred for twodays at 55° C. under inert atmosphere. Isolation and purification of theproduct as described for the preparation of (2.4), yielded (3.1a) (4.2g, 56%) UV (MeOH) λ_(max) 324 nm; ¹H-NMR (d₆-DMSO) δ(ppm) 10.07 (s, 1 H,exchangeable, H—NH—COCF₃), 8.78 (s, 1 H, H-6), 6.63 (s, 1 H,H—CH═C(O)CH₂), 6.18 (t, J=6.55 Hz, 1 H, H-1′), 5.00-5.3 (bm, 2H,exchangeable, 2×OH), 4.48 (m, 2H, H—CH₂—NH), 4.23 (m, 1 H, H-4′), 3.95(m, 1 H, H-3′), 3.63 (m, 2 H, H-5′), 2.40 (m, 1 H, H-2′_(b)), 2.07 (m, 1H, H-2′_(a))

2,2,2-Trifluoro-N-(3-(2-deoxy-β-D-erythro-pentofuranosyI)furano[2,3-d]pyrimidin-2-one-6-methyl)-acetamide(3.1b)

Triethylamine (0.46 g, 0.63 mL, 4.5 mmol, 1.5 eq) was added to a stirredsolution of (2.1b) (1.11 g, 3 mmol, 1 eq), Pd[P(C₆H₅)₃]₄ (0.35 g, 0.3mmol, 0.1 eq), Cul (0.12 g, 0.6 mmol, 0.2 eq) and2,2,2-trifluoro-N-propargyl-acetamide (0.55 g, 3.6 mmol, 1.2 eq) in dryDMF (25 mL) and the resultant reaction mixture was then stirred for twodays at 55° C. under an inert atmosphere. Isolation and purification ofthe product as described for the preparation of (2.4), yielded (3.1b)(0.62 g, 53%): UV (MeOH) λ_(max) 324 nm; ¹H-NMR (d₆-DMSO) δ(ppm) 10.08(s, 1 H, exchangeable, H—NH—COCF₃), 8.90 (s, 1 H, H-6), 6.63 (s, 1 H,H—CH═C(O)CH₂), 5.87 (d, J=6 Hz, 1 H, H-1′), 5.58 (m, 1 H, exchangeable,H—OH), 5.25 (m, 1 H, exchangeable, H—OH), 5.00 (m, 1 H, exchangeable,H—OH), 4.48 (m, 2 H, H—CH₂—NH), 4.00 (m, 3H, H-4′, 3′, 2′), 3.86 (m, 1H,H-5′_(b)), 3.68 (m, 1 H, H-5′_(a))

2,2,2-Trifluoro-N-(3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one-6-methyl)-acetamide(3.2)

The nucleoside (3.1a) (1 g, 2.7 mmol, 1 eq) was dissolved intriethylphosphate (50 mL) and the solution cooled to 0° C. Phosphorusoxychloride (0.163 g, 0.37 mL, 4 mmol) was added dropwise and thereaction mixture stirred at 0° C. under inert atmosphere for threehours. TLC on silica gel (i-Pr—OH/NH₄OH/H₂O-6/3/1) showed reactioncompletion to the monophosphate stage. Tributylamine (2.5 g, 3.25 mL,13.5 mmol, 5 eq) and di-tributylammonium pyrophosphate 0.5M solution indry DMF (27 mL, 13.5 mmol, 5 eq) were simultaneously added at 0° C. Theresulting reaction mixture was stirred at 0° C. for five minutes thenten minutes at room temperature before quenching with 1 M triethylaminebicarbonate (TEAB) buffer (100 mL) and leaving to stir overnight at roomtemperature. After solvent removal, the reaction mixture was dissolvedin water and applied to a SEPHADEX resin (1 L column). Elution with 4 LTEAB buffer in gradient mode from 0.05 M TEAB to 1 M TEAB afforded(3.2). The product was further purified on HPLC using a reverse phaseC18 column using 0.1 mTEAB and 25% MeCN in 0.1M TEAB as eluants over a30 min gradient (0.3 g, 14%): UV (H₂O) λ_(max) 324 nm; ³¹P-NMR (D₂O)δ(ppm) −6.00 (d, J=21.06 Hz), −12.50 (d, J=19.53 Hz), −22.2 (t, J=20.45Hz)

6-Methylamine-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)(3.3a)

Hydrolysis of (3.2) (10.9 mg, 3 mL sol 4.423 mM in water) with 2.5 Msodium carbonate buffer (3 mL) at room temperature for three hours andpurification of the crude product on HPLC using a C18 column and thesame gradient system as above, afforded (3.3a) (5.9 mg, 82%): UV (H₂O)λ_(max) 324 nm; ³¹P-NMR (D₂O) δ(ppm) −4.00 (d, J=21.06 Hz), −10.20 (d,J=19.53 Hz), −19.50 (t, J=20.45 Hz)

5-Carboxyfluoresceinyl-(6-methylamine-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)(3.3b)

5-Carboxyfluorescein-N-hydroxysuccinimide ester (FAM-NHS ester) (1.7 mg,1.25 eq) dissolved in 0.5 mL dry DMF was added to (3.2) (0.84 mg, 14.8mM, 1 eq) dissolved in 0.25 M sodium carbonate/sodium bicarbonate buffer(1 mL) at pH=8.5 and the resulting mixture stirred at room temperaturefor three hours. TLC on silica gel (i-Pr—OH/NH₄OH/H₂O -6/3/1) showedreaction completion. Purification on a short plug of a silicagel columnfollowed by reverse phase HPLC on C18 column afforded (3.3b). UV (H₂O)λ_(max) 324 nm 493 nm

EXAMPLE 4 Synthesis of6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one(4.3a) and5-FAM-(6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)(4.3b)

2,2,2-Trifluoro-(6-methyl-N-(6-amino)-caproamide-3-(2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)-acetamide(4.1)

Treatment of (3.1a) (0.37 g, 1 mmol, 1 eq) with 2.5 M sodium carbonatebuffer (10 mL) for three hours followed by addition of2,2,2-Trifluoro-N-(6-amino-N-Hydroxysuccinimide caproate)-acetamide(0.48 g, 1.5 mmol, 1.5 eq) dissolved in 3 mL of dry DMF, afforded afterchromatography on silicagel (15% methanol/chloroform as eluent) thedesired compound (4.1) (0.27 g, 55%): UV (MeOH) λ_(max) 324 nm; ¹H-NMR(d₆-DMSO) δ(ppm) 9.37 (bs, 1 H, exchangeable, H—NH—COCF₃), 8.74 (s, 1 H,H-6), 8.33 (s, 1 H, exchangeable, H—CH₂NH—COCH₂), 6.50 (s, 1 H,H—CH═C(O)CH₂), 6.12 (t, J=6.45 Hz, 1H, H-1′), 5.27 (m, 1 H,exchangeable, H—OH, 5.11 (m, 1 H, exchangeable, H—OH), 4.21 (m, 3 H,H-3′, OCCH₂NHCO), 3.88 (m, 1 H, H4′), 3.65 (m, 2 H, H-5′), 3.16 (m, 2 H,H—CH₂NHCOCF₃), 2.35 (m, 1 H, H-2′_(b)), 2.15 (t, J=7.27 Hz, 2 H,H—CH₂CONH), 2.00 (m, 1 H, H-2′_(a)), 1.5 (m, 4 H, H—CH₂CH₂CH₂CH₂CH₂),1.26 (m, 2 H, H—CH₂CH₂CH₂CH₂CH₂)

2,2,2-Trifluoro-N-(6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-a]pyrimidin-2-one)-acetamide(4.2)

This compound was prepared from (4.1) (0.169 g, 0.35 mmol, 1 eq) in asimilar manner as (3.2) to afford (4.2) (70 mg, 22%): UV (H₂O) λ_(max)324 nm; ³¹P-NMR (D₂O) δ(ppm) −4.00 (d, J=21.06 Hz), −10.20 (d, J=19.53Hz), −19.50 (t, J=20.45 Hz)

6-Methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one(4.3a)

This compound was prepared from (4.2a) (20 mg, 21.5 mM, 1 eq) in asimilar manner as (3.3a) to afford (4.3a) (15 mg, 84%): UV (H₂O) λ_(max)324 nm; ³¹P-NMR (D₂O) δ(ppm) −4.00 (d, J=21.06 Hz), −10.20 (d, J=19.53Hz), −19.50 (t, J=20.45 Hz)

5-FAM-(6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)(4.3b)

This compound was prepared from (4.2) (1.7 mg, 20.3 mM, 1 eq) in asimilar manner as (3.3b) to afford (4.3b) (0.91 mM, 5%): UV (H₂O)λ_(max) 324 nm 493.8 nm

EXAMPLE 5

Synthesis of2,2,2-Trifluoro-N-(3-(2-deoxy-β-D-erythro-pentofuranosyl)furano-pyrimidin-2-one-6-methyl)-acetamide(5.1)

2,2,2-Trifluoro-N-(3-(2-deoxy-β-D-erythro-pentofuranosyl)5,6-dihydro-furano-pyrimidin-2-one-6-methyl)-acetamide (5.1)

A solution of (3.1a) (0.19 g, 0.5 mmol) in methanol (50 mL) and 5%Pd/Chydrogenation catalyst (0.19 g) were loaded into a Parr hydrogenationapparatus and stirred under hydrogen atmosphere (25 psi) for two hours.TLC on silica gel (15% methanol/chloroform as eluent) showed reactioncompletion. The reaction mixture was filtered and the catalyst washedthree times (50 mL) with hot methanol. Solvent evaporation afforded(5.1) as a diastereomeric mixture (0.16 g, 85%). UV (MeOH) λmax 271 nm;¹H-NMR (d₆-DMSO) δ(ppm) 9.80 (bs, 1H, exchangeable, NH), 9.4 (bs, 1H,exchangeable), 8.18 (s, 1 H), 7.70 (s, 1H) 6.18(m, 2H), (4.80-5.40 (m,4H, exchangeable), 4.20 (m, 2 H), 3.80 (m, 2H), 3.40 (m, 2 H), 3.40,3.25 (m, 4H), 2.80 (m, 1H), 1.90-2.4 and 1.70 (m, 5H)

EXAMPLE 6

Synthesis of6-Butyronitrile-3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one(6.1)

6-Butyronitrile-3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one(6.1)

A solution of (2.1) (0.5 g, 1.24 mmol) in concentrated ammoniumhydroxide (25 mL) was stirred overnight. TLC (30% methanol/chloroform)showed the reaction was complete. Purification on silicagel by flashchromatography (15-25% methanol/chloroform) afforded (6.1) (0.324 g,82.8%) UV (MeOH) λ_(max) 335 nm; ¹H-NMR (d₆-DMSO) δ(ppm) 11.17 (bs, 1H,exchangeable, NH), 8.55 (s, 1 H, H-6), 6.25 (t, J=6.44 Hz, 1 H, H-1′),6.00 (s, 1 H, CH═C(NH)CH₂), 5.25 (m, 1H, exchangeable, H—OH), 5.10 (m, 1H, exchangeable, H—OH), 4.28 (m, 1 H, H-3′), 3.90 (m, 1 H, H4′), 3.68(m, 2 H, H-5′), 2.69 (t, J=, 2H, H—HC═C(O)CH₂CH₂), 2.53 (t, 2 H,H—CH₂CH₂CN), 2.38 (m, 1 H, H-2′_(b)), 2.00 (m, 1 H, H-2′_(a))

EXAMPLE 7

Synthesis of2,2,2-Trifluoro-N-(3-(β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one-6-methyl)-acetamide(7.1b)

2,2,2-Trifluoro-N-(3-(β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one-6-methyl)-acetamide(7.1b)

Overnight treatment of (3.1) (0.37 g, 1 mmol, 1 eq) with concentratedammonium hydroxide solution (10 mL) at room temperature afforded aftersolvent evaporation compound (7.1a). Ethyl trifluoroacetate (1 mL) andtriethylamine (0.1 mL) were added to a methanolic solution of crude(7.1a) and the reaction mixture stirred for 4 hours at room temperature.Solvent removal and flash chromatography with 15% methanol/chloroform aseluent afforded (7.1b) (0.286 g, 76%): UV (MeOH) λ_(max) 335 nm; ¹H-NMR(d₆-DMSO) δ(ppm) 11.50 (bs, 1 H, exchangeable, H—NH-pyrrol ring), 10.18(bs, 1 H, exchangeable, H—NH—COCF₃), 8.70 (s, 1 H, H-6), 6.25 (t, J=6.55Hz, 1 H, H-1′), 6.08 (s, 1 H, H—CH═C(NH)CH₂),), 5.25 (m, 1H,exchangeable, H—OH), 5.10 (m, 1 H, exchangeable, H—OH), 4.27 (m, 3 H,H—═C(NH)CH₂COCF₃, and 4′), 3.90 (m, 1 H, H-3′), 3.68 (m, 2H, H-5′), 2.38(m, 1 H, H-2′_(b)), 2.07 (m, 1 H, H-2′_(a))

EXAMPLE 8

Synthesis of6-methylamine-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one)(8.1)

6-methylamine-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one)(8.1)

Treatment of (3.2) (10.9 mg, 3 mL sol 4.423 mM in water) withconcentrated ammonium hydroxide solution (3 mL) at room temperature forthree hours and purification on HPLC (C₁₈) of the crude product,afforded (8.1) (0.6 mg, 8%): UV (H₂O) λ_(max) 268 nm 335 nm

EXAMPLE 9

Synthesis of2,2,2-Trifluoro-(6-methyl-N-(6-amino)-caproamide-3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one)-acetamide(9.1)

2,2,2-Trifluoro-(6-methyl-N-(6-amino)-caproamide-3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one)-acetamide(9.1)

This compound was prepared from (4.1a) (0.1 g, 0.2 mmol, 1 eq) in asimilar manner as (7.1b) to afford (9.1) (0.074 g, 76%): UV (MeOH)λ_(max) 335 nm; ¹H-NMR (d₆-DMSO) δ(ppm) 9.37 (bs, 1 H, exchangeable,H—NH—COCF₃), 9.36 (bs, 1 H, exchangeable, H—N═C—NH—C(CH₂)═CH), 8.59 (s,1 H, H-6), 8.28 (s, 1 H, exchangeable, H—CH₂NH—COCH₂), 6.25 (dd, J=8.10Hz, 5.4 Hz, 1 H, H-1′), 6.02 (s, 1 H, H—CH═C(NH)CH₂), 5.27 (m, 1 H,exchangeable, H—OH), 5.11 (m, 1 H, exchangeable, H—OH), 4.21 (m, 3 H,H-3′, OCCH₂NHCO), 3.88 (m, 1 H, H-4′), 3.65 (m, 2 H, H-5′), 3.16 (m, 2H, H—CH₂NHCOCF₃), 2.35 (m, 1 H, H-2′_(b)), 2.15 (t, J=7.27 Hz, 2 H,H—CH₂CONH), 2.00 (m, 1 H, H-2′_(a)), 1.5 (m, 4 H, H—CH₂CH₂CH₂CH₂CH₂),1.26 (m, 2 H, H—CH₂CH₂CH₂CH₂CH₂)

EXAMPLE 10

Synthesis of dye conjugates of6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one(10.1a)

6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one(10.1a)

This compound was prepared from (4.2) (30 mg, 35.8 mM, 1 eq) in asimilar manner as (8.1a) to afford (10.1a) (20 mg, 24.3 mM, 68%): UV(H₂O) λ_(max) 335 nm

5-FAM-(6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one)(10.1b)

This compound was prepared from (10.1a) (6.5 mg, 7.8 mM, 1 eq) in asimilar manner as (8.1b) to afford (10.1b) (1.88 mM, 24%): UV (H₂O)λ_(max) 335 nm 494 nm

Cy5-(6-methyl-N-(6-amino)-caproamide-3-(triphospho-2-2deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one(10.1c)

This compound was prepared from (10.1a) in a similar manner as (8.1b) toafford (10.1c): UV (H₂O) λ_(max) 355 nm, 648 nm

EXAMPLE 11

1-(2′-deoxy-5′-triphospho-β-D-ribofuranosyl)piperidino[2,3-d]pyrimidine-2(1H)-one(1.6),6-methylamine-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)(3.3a) and5-FAM-(6-methylamine-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)(3.3b),6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one(4.3a) and5-FAM-(6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)furano[2,3-d]pyrimidin-2-one)(4.3b),6-methylamine-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one)(8.1),6-methyl-N-(6-amino)-caproamide-3-(5-triphospho-2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one(10.1a), and the FAM and Cy5 of (10.1a, e.g. 10.1b and 10.1c) assubstrates for terminal deoxynucleotidyl transferase. In order to testthe ability of compounds (1.6), (3.3a), (3.3b), (4.3a), (4.3b), (8.1),(10.1a), (10.1b) and (10.1c) to be accepted by terminal deoxynucleotidyltransferase as a substrate, an oligonucleotide tailing reaction wasperformed.

A 15 mer primer (sequence: 5′ TGC ATG TGC TGG AGA 3′) and 8 to 32 baseoligonucleotide markers were 5′ end labelled with [γ³³P] ATP and T4polynucleotide kinase. Reactions were boiled for 5 minutes afterlabelling to remove any PNK activity. Four picomoles of the labelledprimer, 25 U terminal deoxynucleotidyl transferase and 32 μM dATP, dCTP,dGTP, dTTP or compounds (1.6), (3.3a), (3.3b), (4.3a), (4.3b), (8.1),(10.1a), (10.1b) and (10.1c) were incubated in 25 μl 100 mM cacodylatebuffer pH7.2, 2 mM CoCl₂ and 0.2 mM 2-mercaptoethanol for 90 minutes at37° C. The reactions were stopped by the addition of formamide stopsolution and the reaction products run on a 19% polyacrylamide 7M ureagel with the labelled markers. Autoradiography using Biomax film wascarried out on the dry gel.

The results showed that the natural nucleotides gave 3′ tails in theregion of 50 to 120 bases. The order of tail size being dTTP, dATP, dCTPthen dGTP. Compound (1.6) gave 3′ tails equivalent to those produced bydCTP at the same concentration. Compound (10.1a) gave a tail of ˜7-8bases. Compound (10.1b) gave tails in the 18-24 base range. Compound(10.1c) gave tails greater than 30 bases in length, which is surprisingcompared to the tails produced in the presence of fluor labelled naturaldNTP's. Compound (8.1) produced a tail 2-3 bases in length. Compound(3.3a) gave a tail 2-3 bases in length. Compound (3.3b) produced a tail2-3 bases in length. Compound (4.3a) gave a tail 7-9 bases in length.Compound (4.3b) gave a tail 9-14 bases in length. This shows that thecompounds of the invention are all substrates for terminaldeoxynucleotidyl transferase and can therefore be incorporated intonucleic acids as a means of labelling or adding a new highly reactivefunctional group.

EXAMPLE 12

Primer Extension Assays to Study Incorporation of Nucleotides (10.1a),(10.1b) and (10.1c) by DNA Polymerases

A primer extension assay was used to evaluate compounds (10.1a), (10.1b)and (10.1c) as substrates for exonuclease free Klenow fragment of DNApolymerase I (EFK). The assay used a ³³P 5′ end labelled 15 mer primerhybridised to a 24 mer template. The sequences of the primer andtemplate are:

Primer 5′ TGCATGTGCTGGAGA 3′ Template 3′ACGTACACGACCTCTGCCTTGCTA 5′

One picomole ³³P labelled primer was hybridised to 2 picomoles oftemplate in ×2 Klenow buffer. To this was added either 20 μM dCTP or 20μM (10.1a) or 20 μM (10.1b) or 20 μM (10.1c) or mixtures of dCTP andcompound (10.1a) or compound (10.1b) or compound (10.1c) keeping thetotal nucleotide concentration fixed at 20 μM. One unit EFK and 2 mUinorganic pyrophosphatase were used per reaction. Primer alone, primerplus template plus enzyme controls were also carried out. The reactionswere incubated at 37° C. for 3 minutes. Reactions were then stopped bythe addition of formamide/EDTA stop solution. Reaction products wereseparated on a 19% polyacrylamide 7M urea gel and the product fragmentssized by comparison with a ³³P labelled 8 to 32 base oligonucleotideladder after exposure to Kodak Biomax autoradiography film.

The autoradiogram showed that all three compounds were substrates forthe polymerase being incorporated in place of dCTP. Compound (10.1a) wasa surprisingly good substrate compared to the results obtained withsimilarly functionalised dCTP analogues like 5-allylaminocaproamidodCTP. This result shows that the analogues of this invention are usefulin labelling DNA and also in introducing a highly reactive functionalityinto the DNA which would not normally be present.

EXAMPLE 13

Primer Extension Assays to Study Incorporation of Compounds (3.3a),(3.3b), (4.3a),(4.3b) and (8.1) by DNA Polymerases

A primer extension assay was used to evaluate compounds (3.3a), (3.3b),(4.3a),(4.3b) and (8.1) as substrates for exonuclease free Klenowfragment of DNA polymerase I (EFK). The assay used a ³³P 5′ end labelled15 mer primer hybridised to one of two 24 mer templates. The sequencesof the primer and templates are:

Primer 5′ TGCATGTGCTGGAGA 3′ Template 1 3′ ACGTACACGACCTCTACCTTGCTA 5′Template 2 3′ ACGTACACGACCTCTGAACTAGTC 5′

One picomole ³³P labelled primer was hybridised to 2 picomoles oftemplate in ×2 Klenow buffer. To this was added either 20 μM dCTP or 20μM (3.3a) or 20 μM (3.3b) or 20 μM (4.3a) or 20 μM (4.3b) or 20 μM (8.1)or mixtures of dCTP and compound (3.3a) or (3.3b) or (4.3a) or (4.3b) or(8.1) keeping the total nucleotide concentration fixed at 20 μM. Oneunit EFK and 2 mU inorganic pyrophosphatase were used per reaction.Primer alone, primer plus template plus enzyme controls were alsocarried out. The reactions were incubated at 37° C. for 3 minutes.Reactions were then stopped by the addition of formamide/EDTA stopsolution. Reaction products were separated on a 19% polyacrylamide 7Murea gel and the product fragments sized by comparison with a ³³Plabelled 8 to 32 base oligonucleotide ladder after exposure to KodakBiomax autoradiography film.

The autoradiograms showed that compound (4.3b) was only incorporated inreactions using Template 2, thus indicating its preference for replacingdCTP. Compound (3.3b) was not a substrate for the polymerase usingeither of the Templates.

Compound (8.1) was a substrate for the polymerase being able tosubstitute for dCTP. Again this is very surprising as5-allylaminocaproamide dCTP did not show any incorporation under similarassay conditions.

Compound (4.3a) was also a substrate for the polymerase again being ableto replace dCTP. However, compound (3.3a) was not a substrate for thepolymerase. This highlights the surprising nature of the resultsobtained with compounds (4.3a), (8.1) and (10.1a). In each case theanalogue with the longer linker arm is the better substrate for thepolymerase i.e. (10.1a) is a better substrate than (8.1), (4.3a) is abetter substrate than (3.3a), which in fact is not a substrate for thepolymerase under the conditions tested.

EXAMPLE 14

Primer Extension Assays to Study1-(2′-deoxy-5′-triphospho-β-D-ribofuranosyl)piperidino[2,3-d]pyrimidine-2(1H)-one(1.6) Incorporation by DNA Polymerases

A primer extension assay was used to evaluate compound (1.6) as asubstrate for exonuclease free Klenow fragment of DNA polymerase I(EFK). The assay used a ³³P 5′ end labelled 15 mer primer hybridised toa 24 mer template. The sequences of the primer and template are:

Primer 5′ TGCATGTGCTGGAGA 3′ Template 3′ ACGTACACGACCTCTGAACTAGTC 5′

One picomole ³³P labelled primer was hybridised to 2 picomoles oftemplate in ×2 Klenow buffer. To this was added either 4 μM dNTPαS or 80μM (1.6) or a mixture of 4 μM dNTPαS 80 μM (1.6). One unit EFK and 2 mUinorganic pyrophosphatase were used per reaction. Primer alone, primerplus template plus enzyme controls were also carried out. The reactionswere incubated at 37° C. for 3 minutes. Reactions were then stopped bythe addition of formamide I EDTA stop solution. Reaction products wereseparated on a 19% polyacrylamide 7M urea gel and the product fragmentssized by comparison with a ³³P labelled 8 to 32 base oligonucleotideladder after exposure to Kodak Biomax autoradiography film.

This showed that (1.6) was a substrate for EFK and that it wasefficiently incorporated in place of dCTP.

EXAMPLE 15

Incorporation of Compound (10.1a) into cDNA and PCR Products

1^(st) Strand cDNA Synthesis

Incorporation of compound (10.1a) into 1^(st) strand cDNA was carriedout using a balance of dCTP such that the total concentration of (10.1a)and dCTP was equivalent to each of the other three dNTP concentrations.

(10.1a) was used at three different percentages

1) 50% (10.1a)

2) 75% (10.1a)

3) 100% (10.1a)

Nucleotide Solutions (10.1a) level 50% 75% 100% 10 mM dATP 1 μl   1 μl 1μl 10 mM dGTP 1 μl   1 μl 1 μl 10 mM dTTP 1 μl   1 μl 1 μl  5 mM dCTP 1μl 0.5 μl 0 μl  5 mM (10.1a) 1 μl 0.5 μl 2 μl

These reactions were compared with reactions including Cy5 labelleddCTP. All reactions were monitored by carrying out the reaction induplicate and monitoring one of the duplicates with a spike of ³³P-dATP.

Reaction Matrix

Sample

1. 50% (10.1a)

2. 50% (10.1a)+³³P-dATP

3. 75% (10.1a)

4. 75% (10.1a)+³³P-dATP

5. 100% (10.1a)

6. 100% (10.1a)+³³P-dATP

Cy5 dCTP

Cy5 dCTP+³³P-dATP

1^(st) Strand cDNA Synthesis

To a reaction tube add:

1 μg mRNA

1 μg anchored dT₍₂₅₎ primer

10 μl water

Heat at 70° C. for 5 minutes

Incubate at room temp. for 10 minutes

Transfer tube to ice and add

4 μl buffer

4 μl NaPPi

1 μl HPRI

1 μl dNTP mix (50%, 75% or 100% (10.1a)) or Cy5 dCTP

1 μl ³³P-dATP-active samples only

1 μl AMV reverse transcriptase 20 U/μl

Incubate at 42° C. for 2½ hours

Store on ice

Alkaline Hydrolysis

Heat reactions at 94° C. for 3 minutes

Add 1 μl of 5M NaOH

Incubate at 37° C. for 10 minutes

Add 1 μl of 5M HCl

Add 5 μl of 1M Tris.HCl, pH 6.8

Mix and centrifuge briefly.

The cDNA reactions were monitored by TLC as follows

1 μl of sample was spotted onto PEI cellulose TLC plates and run in1.25M Potassium phosphate buffer

TLC results (10.1a) ³³P-dATP Incorporation  50% 23.2%  75% 16.5% 100%11.5% Standard cDNA 15.9%

Samples were then purified by Qiagen column and eluted in 50 μl ofwater. The active cDNA products were analysed on a 6% polyacrylamide gel

8 μl of sample+3 μl of loading dye

Run at 35 mV for 1½ hours

The gel was then covered in Saran wrap and exposed to phosphorscreen for1 hour. Phosphorscreen analysis was carried out on the MolecularDynamics Storm 860 instrument.

Results

6% polyacrylamide gel analysis showed that cDNA products had been madein all reactions.

PCR Reactions

Incorporation of (10.1a) into arabidopsis DNA by PCR was carried out asfollows:

The (10.1a) was used in the PCR at various percentages with a balance ofdCTP such that the total concentration of (10.1a) and dCTP wasequivalent to each of the other three base dNTP's. The percentages of(10.1a) used were:

%(10.1a)

0%

5%

10%

20%

30%

40%

50%

100%

Compound (10.1a) PCR conditions

To each reaction tube was added

Arabidopsis thaliana template DNA (100 pg/μl)   5 μl 10X PCR reactionbuffer   5 μl T7 forward primer (5 μM)   5 μl T3 reverse primer (5 μM)  5 μl dATP (4 mM) 2.5 μl X% (10.1a) (4 mM) 2.5 μl dGTP (4 mM) 2.5 μldTTP (4 mM) 2.5 μl Thermus aquaticus DNA polymerase (5 units/μl) 0.5 μlwater 19.5 μl  Total  50 μl

The reaction mix was covered with two to three drops of mineral.

The reaction tubes were placed in the PCR thermocycling block.

PCR Thermocycling:—Carried Out in a Perkin Elmer DNA Thermal Cycler 480

35 cycles of 94° C. 45 seconds 45° C. 45 seconds 72° C. 2 minutesFollowed by 72° C. 8 minutes  4° C. soak

After PCR, the samples were purified by Qiagen column and analysed by 1%agarose gel.

10 μl sample+1 μl Vistra green

Run at 150V for 1½ hours

Results

Agarose gel analysis showed that all levels of (10.1a) resulted in theformation of PCR product EXCEPT for the 100% (10.1a) level.

Attachment of Cy5 NHS-Ester to (10.1a) Incorporated into cDNA and PCRProduct

Cy5 NHS-Ester was coupled to (10.1a) bases incorporated into DNA by theabove methods as follows:

Cy5 NHS-Ester (Amersham Pharmacia Biotech)

Q15108 Lot: 973181

1 vial dissolved in 1 ml of dry DMF

Labelling reaction

20 μl DNA

5 μl Cy5 NHS-Ester

25 μl sodium bicarbonate buffer, pH 9.3

50 μl Total volume

Labelling Matrix Tube % (10.1a) Inc. Method 1 + 2  0 PCR 3 + 4  5 PCR5 + 6  10 PCR 7 + 8  20 PCR  9 + 10  30 PCR 11 + 12  40 PCR 13 + 14  50PCR 15 + 16  75 cDNA 17 + 18 100 cDNA 19 + 20 M13- standard DNA to checkfor non-specific labelling

The dye was allowed to react with the (10.1a) groups in the DNA for 16hours (overnight)

The following day, 2 μl of one of each duplicate was analysed on silicagel TLC.

TLC system

Silica gel—normal phase, 740238909 (Merck)

Solvent—60% IPA (iso propyl alcohol)

20% NH₄OH

20% Water

The samples were spotted and run for 1 hour. The TLC sheet was thendried and analysed on the Molecular Dynamics Storm 860 instrument.

Cy5 NHS-Ester Labelling Results

The scan showed evidence of Cy5 labelling in tubes 1-18, but not tubes19-20, indicating that the NHS-Ester has coupled with the (10.1a) groupsincorporated into the DNA. Increasing Cy5 labelling was seen withincreasing percentage of compound (10.1a) in the reaction. This clearlyshows the ability of this analogue to introduce functionality into theDNA. This functionality could be used for labelling as above or forattachment to a solid support.

What is claimed is:
 1. A compound having the structure

where X=O or NH or S Y=N or CHR⁶ or CR⁶ or CO W=N or NR⁶ or CHR⁶ or CR⁶or S or CO n=2 each R⁶ is independently H or alkyl or alkenyl or alkoxyor aryl or a reporter moiety, if Y or W=N or CR⁶, then a double bond ispresent between the Y and W or between the W and W, and Q is selectedfrom the group consisting of H, a sugar, a sugar analogue, a nucleicacid backbone, and backbone analogue provided that (i) when X is NH andW is CHR⁶ or CR⁶, and Y is CO, then at least one R⁶ is a reportermoiety, (ii) when W is S, then W_(n) is —CHR⁶—S— or ═CR⁶—S—, (iii) whenX is NH and Y is CHR⁶ or CR⁶, then at least one R⁶ is a reporter moietythat is selected from the group consisting of a reactive group, signalmoiety, and solid surface joined to the remainder of the molecule by alinker of at least 3 chain atoms, and (iv) when X is O and Y is CHR⁶ andW is CHR⁶ or S, then at least one R⁶ is a reporter moiety.
 2. Thecompound of claim 1 wherein the compound is (a) a nucleoside ornucleoside analogue wherein Q is

 where Z is O R¹, R², R³ and R⁴ are the same or different and each is Hor OH R⁵ is mono-, di-, or tri-phosphate, or one of R² and R⁵ is aphosphoramidite or other group for incorporation in a polynucleotidechain; or (b) a polynucleotide wherein Q is a nucleic acid backboneselected from the group consisting of sugar-phosphate repeats, modifiedsugar-phosphate repeats (LNA), or a backbone analogue such as peptide orpolyamide nucleic acid (PNA).
 3. A compound of claim 1 wherein thestructure is selected from the group consisting of


4. The compound of claim 3, wherein a reporter moiety is present.
 5. Thecompound of claim 4, wherein the reporter moiety is a signal moiety. 6.The compound of claim 4, wherein the reporter moiety is a reactive groupor signal moiety or solid surface joined to the remainder of themolecule by a linker of at least 3 chain atoms.
 7. The compound of claim3, wherein R⁵ is triphosphate.
 8. The compound of claim 3, wherein oneof R² and R⁵ is selected from phosphoramidite and H-phosphonate.
 9. Apolynucleotide comprising at least one residue of the compound ofclaim
 1. 10. The polynucleotide of claim 9, wherein the polynucleotideis DNA or RNA.
 11. A chain extension method which comprises reacting apolynucleotide with a nucleoside triphosphate analogue of claim 1 in thepresence of a polymerase or a terminal deoxynucleotidyl transferaseenzyme.
 12. A method of detecting the polynucleotide of claim 9, whichmethod comprises using for detection an antibody which binds to a basecomponent selected from the group consisting of structure (1), structure(2), structure (3), and structure (4).
 13. A method of making cDNA whichcomprises incubating an RNA template with a monomer mixture including anucleotide analogue as claimed in claim 1 in the presence of a reversetranscriptase.
 14. A method of amplifying a polynucleotide by PCR whichmethod comprises using a monomer mixture including a nucleotide analogueas claimed in claim
 1. 15. A method of reducing compression artefacts innucleic acid sequencing, comprising (a) incorporating at least onenucleoside analogue of claim 1 into polynucleotides using a polymerase;and (b) separating polynucleotides created by the polymerase byelectrophoresis, wherein incorporation of the nucleoside analogue intoone or more polynucleotides reduces detection of compression artefacts.16. A method of detecting a residue of the compound of claim 1, themethod comprising analyzing by mass spectrometry.